PEX3 promotes regenerative repair after myocardial injury in mice through facilitating plasma membrane localization of ITGB3

The peroxisome is a versatile organelle that performs diverse metabolic functions. PEX3, a critical regulator of the peroxisome, participates in various biological processes associated with the peroxisome. Whether PEX3 is involved in peroxisome-related redox homeostasis and myocardial regenerative repair remains elusive. We investigate that cardiomyocyte-specific PEX3 knockout (Pex3-KO) results in an imbalance of redox homeostasis and disrupts the endogenous proliferation/development at different times and spatial locations. Using Pex3-KO mice and myocardium-targeted intervention approaches, the effects of PEX3 on myocardial regenerative repair during both physiological and pathological stages are explored. Mechanistically, lipid metabolomics reveals that PEX3 promotes myocardial regenerative repair by affecting plasmalogen metabolism. Further, we find that PEX3-regulated plasmalogen activates the AKT/GSK3β signaling pathway via the plasma membrane localization of ITGB3. Our study indicates that PEX3 may represent a novel therapeutic target for myocardial regenerative repair following injury.

The manuscript by Sun et al. aims to identify the role of the peroxisome regulator, PEX3, in myocardial regeneration.The authors observed an increase in RNA and protein levels of PEX3 during regeneration.They found that deletion of pex3 reduces postnatal cardiomyocyte proliferation, while overexpression of Pex3 promotes this proliferation in vitro.Importantly, they demonstrate that Pex3 deletion impedes cardiomyocyte proliferation and neonatal heart regeneration following apical resection.Additionally, in vivo overexpression of Pex3 via AAV9 enhances adult cardiomyocyte proliferation, decreases scar size, and improves cardiac function post-myocardial infarction (MI).To explore the mechanisms behind Pex3's role in myocardial regeneration, the authors conducted lipidomic analyses and discovered a reduction in plasmalogen levels in Pex3 knockout mice.Supplementation of plasmalogen improved the regenerative response in these mice.Further investigations revealed that plasmalogen regulates the localization of ITGB3 in the plasma membrane, which in turn mediates AKT signaling involved in the regenerative effects of Pex3.Therefore, the authors conclude that Pex3 facilitates cardiomyocyte proliferation and regeneration through the modulation of plasmalogen metabolism, which affects ITGB3 and AKT signaling.This is a comprehensive study that provides new insights into the role of Pex3 and the peroxisome during heart regeneration.A few points can be addressed which would strengthen the conclusions of this study: • Can the authors administer AAV9-cTnT-PEX3 systemically in adult mice at 3 days post-MI after confirmation of infarct sizes to fully establish its impact on regeneration.Additionally, the systemic injection might promote higher levels of cardiomyocyte proliferation throughout the myocardium rather than the border zone only.
• For plasmalogen supplementation in Figure 6 and 7, confirmation of infarct sizes and cardiac function at an earlier timepoint post-MI to ensure equal infarction is necessary, as it is difficult to interpret this data as regeneration rather than protection or surgical variability.While the current adult data is promising, it doesn't distinguish between protection or regeneration.
Reviewer #2 (Remarks to the Author): The authors present a large set of experiments and data to help describe how the peroxisome is involved during regeneration.This has so far not been well understood and the manuscript adds an interesting layer to the complicated yet coordinated events following heart injury and will be of interest to the wider scientific community.However there are a number of points the reviewer would like to be experimentally addressed.Furthermore the text is dense, there is a lot of main figures and the ordering of both the main and supplemental figures requires the reader to jump between figures making it difficult to follow the story line.Some editing (rearranging figures, moving main figures to supplement or removing entirely) will be beneficial for helping the reader follow the manuscript.

Major
-while the increase in PMP70 and PEX3 at the BZ in neonates supports the hypothesis, the decrease in PMP70 and PEX3 in adult IZ and BZ is confounding.Wouldn't the authors predict it to be unchanged?Does this suggest that in the adult context, there is a different feedback in peroxisome regulation when compared to the neonates and if so how is this regulated?-the author states an increase in PMP70 in the immunostainings in supplemental Figure 1E.Please quantify this as it appears there are also PMP70 positive cells in non-cardiomyocytes that might be masking the actual result.The same for the non-cardiomyocyte PEX3 stains in Fig1E-F -figure 1A shows increase in PEX3 but also PEX12.PEX12 is not mentioned at all, how does this fit in with the author's data?-Ad5 PEX3 overexpression suggests it is cardioprotective, while AAV9 PEX3 overexpression suggests it promotes positive regeneration.Are these linked?-I assume AR in line 247 means apical resection?I find it strange that this was the choice to induce heart injury.The neonatal LAD model (which is later used in the manuscript in adults) would be more appropriate to conduct the experiments since this model better induces the hypoxia/ischemia conditions seen following MI.
-increased fibrosis in Pex3-KO mentioned from line 263 onwards needs to be explored more.How is PEX3 and to a broader degree peroxisomes mechanistically affecting fibrosis?Are the fibroblasts being activated?How do peroxisomes interact with fibroblasts when the model presented is a cardiomyocyte specific KO?Is the increase in fibrosis coming from increased immune cell activity/activation? How this links to the proliferation data is also not clear -similarly, in the PEX3 overexpression in adult MI model, the data suggests the scar is being removed.Is this really the case?If so how is this achieved given that AAV9 delivery is generally cardiomyocyte specific.Are other cell types such as fibroblasts or immune cells involved?-echo analysis to assess function at 28dpi is line 294 is rather early to state a functional recovery, particularly when the scar is still present.Please provide later timepoints if the authors want to keep this statement or tone down -it is unclear if the authors are trying to establish a direct link between proliferation and fibrosis following PEX3 modulation.The text in some places suggests it is while not in other places.Please be clearer and if a direct link is suggested, please provide the data to support these claims.
-ITGB3 appears to be downstream of PEX3, what do the peroxisomes look like in ITGB3 overexpression/knockdown?Is the PEX3/ITGB3 interaction direct or only indirect via plasmalogens?-is peroxisome density (eg.PMP70 levels) modulated when ITGB3 is manipulated?Minor -the use of abbreviations and not defining what it actually is makes it difficult to follow.For example dps, dpr, AR, DCF, MOI are examples of abbreviations used but I actually have no idea what the authors are referring to -Please specify more clearly what is P7/6dps, P7/6dpr, P56/6dps/P56/6dpi.These abbreviations were not defined anywhere in the main text and i had to go hunting in an unrelated figure legend to figure out what the authors were talking about.Please rename these abbreviations to something that is more easy for the reader to understand.There is already so much abbreviations for other names in the text that the author's message gets lost -why is P7/6dpr and P56/6dpi in Sup.fig1E and P7/6dpr and P56/6dpi in Sup.Fig 1F  -can the authors provide data that the cardiomycoyte populations used for Ad5 transfection are pure and non-cardiomycoytes obtained from the isolations are not affecting the downstream analysis?-can the authors provide data that the AAV9 transfections is targeting only cardiomyocytes?

Responses to the editorial office and reviewers:
Thanks for your advice and we are delighted to have the opportunity to revise our manuscript entitled "PEX3 promotes regenerative repair after myocardial injury through facilitating plasma membrane localization of ITGB3" (Manuscript Number: COMMSBIO-24-0061-T).We are grateful for the thoughtful and helpful comments from the editor and the reviewers on our manuscript.We carefully modified the manuscripts according to the comments.All changes in the manuscript are marked in red font in the revised Manuscript and Supplementary Materials.Below are our point-to-point responses.
Thanks again for your consideration.

Can the authors administer AAV9-cTnT-PEX3 systemically in adult mice at 3 days post-MI after confirmation of infarct sizes to fully establish its impact on regeneration.
Additionally, the systemic injection might promote higher levels of cardiomyocyte proliferation throughout the myocardium rather than the border zone only.
Response: Thanks so much for this important comment.We followed the suggestion and performed additional experiments.We injected AAV9-cTnT-PEX3 via the tail vein into adult mice with comparable LVEF at 3 days after myocardial infarction.We examined the proliferation levels of cardiomyocytes in the infarct border zone as well as in the normal zone, respectively.The results showed that overexpression of PEX3 systemically promotes the proliferation of cardiomyocytes in the normal zone and border zone (Supplementary Figure 7C-E).However, whether systemic overexpression of PEX3 has potential ectopic tumourigenicity is still an important work, which will be explored in subsequent studies.The related contents are added in Methods (Page 8, line 198-200), Results (Page 26, line 715-722) and Discussion (Page 36, line 1008-1011) now.6 and 7, confirmation of infarct sizes and cardiac function at an earlier timepoint post-MI to ensure equal infarction is necessary, as it is difficult to interpret this data as regeneration rather than protection or surgical variability.While the current adult data is promising, it doesn't distinguish between protection or regeneration.

For plasmalogen supplementation in Figure
Response: Thanks for the helpful comments.To ensure acceptable homogeneity of the infarction model in experimental animals, we included animals with comparable echocardiography-derived LVEF (Supplementary Figure 9A LVEF: 53.52%±0.8042;Supplementary Figure 11E LVEF: 53.51%± 0.7707) at 1dpi in our study (Supplementary Figure 9A and Supplementary Figure 11E).Protection and repair after the myocardial injury is an ambitious scope that includes myocardial regeneration, inflammation regulation, neovascularisation and interstitial remodelling 1 .In our work, we found that PEX3 mainly promotes cardiomyocyte proliferation to achieve regenerative repair after injury by detecting cardiomyocyte proliferation indexes.We have revised the expression accordingly in the manuscript.1. Uygur A, Lee RT.Mechanisms of Cardiac Regeneration.Dev Cell.2016 Feb 22;36(4):362-74.PMID: 26906733; PMCID: PMC4768311.

Reviewer 2:
The authors present a large set of experiments and data to help describe how the peroxisome is involved during regeneration.This has so far not been well understood and the manuscript adds an interesting layer to the complicated yet coordinated events following heart injury and will be of interest to the wider scientific community.However there are a number of points the reviewer would like to be experimentally addressed.Furthermore the text is dense, there is a lot of main figures and the ordering of both the main and supplemental figures requires the reader to jump between figures making it difficult to follow the story line.Some editing (rearranging figures, moving main figures to supplement or removing entirely) will be beneficial for helping the reader follow the manuscript.Response: Thank you for the helpful comments.In the revised manuscript, we carefully considered all the related issues and made corresponding adjustments, modifications, and supplements to the figures and text.
Major 1. while the increase in PMP70 and PEX3 at the BZ in neonates supports the hypothesis, the decrease in PMP70 and PEX3 in adult IZ and BZ is confounding.Wouldn't the authors predict it to be unchanged?Does this suggest that in the adult context, there is a different feedback in peroxisome regulation when compared to the neonates and if so how is this regulated?
Response: Thank you for the thoughtful comments.We found experimentally that PEX3-related peroxisome function is activated after neonatal myocardial injury, whereas the expression is decreased after adult myocardial injury.So, we think activation of PEX3-related peroxisome function is closely related to the regenerative repair process after myocardial injury.This functional mechanism is preserved in neonatal mice, but lost in adult mice.Peroxisomes, as the prominent effector organelles of reactive oxygen species, play an essential role in the cellular stress response and the regulation of cellular redox homeostasis, and peroxisomes are subjected to mobility regulation to adapt to changing cellular demands and different external environments 2 .In this study, we explored the underlying molecular mechanisms of PEX3, to see if PEX3 could act as a critical factor in the regulation of regenerative repair even after myocardial injury in adult mice.Deciphering the biological processes that dynamically control peroxisomes remains a significant challenge that we will pursue further in future work.The above information is added in the revised manuscript.It reads (Page 36, line 990-1003): "However, some limitations should be noted.We found experimentally that PEX3-related peroxisome function is activated after neonatal myocardial injury, whereas the expression is decreased after adult myocardial injury.So, we think activation of PEX3-related peroxisome function is closely related to the regenerative repair process after myocardial injury.This functional mechanism is preserved in neonatal mice, but lost in adult mice.Peroxisomes, as the prominent effector organelles of reactive oxygen species, play an essential role in the cellular stress response and the regulation of cellular redox homeostasis, and peroxisomes are subjected to mobility regulation to adapt to changing cellular demands and different external environments 1 .In this study, we explored the underlying molecular mechanisms of PEX3, to see if PEX3 could act as a critical factor in the regulation of regenerative repair even after myocardial injury in adult mice.Deciphering the biological processes that dynamically control peroxisomes remains a significant challenge that we will pursue further in future work".1. Di Cara F, Savary S, Kovacs WJ, Kim P, Rachubinski RA.The peroxisome: an up-and-coming organelle in immunometabolism.Trends Cell Biol.2023 Jan;33(1):70-86.Epub 2022 Jul 1. PMID: 35788297.1E.Please quantify this as it appears there are also PMP70 positive cells in non-cardiomyocytes that might be masking the actual result.The same for the non-cardiomyocyte PEX3 stains in Fig1E-F Response: Thanks for pointing out this important issue.As suggested, we quantified the intensity of PMP70 and PEX3 and found that PMP70 or PEX3 was overexpressed after neonatal myocardial injury and decreased after adult myocardial injury (Supplementary Figure 1E and Supplementary Figure 2C).Additional co-localization immunofluorescence experiments were performed, and results showed that PMP70 or PEX3 expression was not enriched in non-cardiomyocytes marked by Vimentin, CD68, and CD31 at P7/6dpr and P56/6dpi (Supplementary Figure 1A-B and Supplementary Figure 2A-B

figure 1A shows increase in PEX3 but also PEX12. PEX12 is not mentioned at all, how does this fit in with the author's data?
Response: Thanks for the helpful comments.Yes, the expressions of PEX3 and PEX12 were both increased after neonatal myocardial injury, and the increase of PEX3 was more significant (Figure 1B).It is known that the most severe peroxisome-deficient phenotype was in yeast PEX3 cells, in which most of the peroxisome matrix and membrane proteins were mislocalised 4 .Whereas PEX12 is a part of genes that encode peroxins involved in matrix protein import.Mutation in PEX12 results in smaller, spherical peroxisomal structures 5 .In polymorphic H.PEX3 cells, only a fraction of the PMPs is present on peroxisomal membrane structures.These proteins are stable relative to other PMPs, which are also incorrectly localized to the cytoplasm 6 .As a result, the different peroxins acting on matrix protein import and peroxisomal membrane biogenesis are often unable to be aligned, and this may behave differently in different model organisms 7 .In general, PEX3 is a prerequisite for peroxisomal regulation and served as a key signaling in this study, the next, we will further explore whether PEX3 is valuable in myocardial regeneration.The above information has been added to the discussion section.It reads (Page 32-33, line 891-904): "We initially found that the expressions of PEX3 and PEX12 were both increased after neonatal myocardial injury, and the increase of PEX3 was more significant.It is known that the most severe peroxisome-deficient phenotype was in yeast PEX3 cells, in which most of the peroxisome matrix and membrane proteins were mislocalized 1 .Whereas PEX12 is a part of genes that encode peroxins involved in matrix protein import.Mutation in PEX12 results in smaller, spherical peroxisomal structures 2 .In polymorphic H.PEX3 cells, only a fraction of the PMPs is present on peroxisomal membrane structures.These proteins are stable relative to other PMPs, which are also incorrectly localized to the cytoplasm 3 .As a result, the different peroxins acting on matrix protein import and peroxisomal membrane biogenesis are often unable to be aligned, and this may behave differently in different model organisms 4 .In general, PEX3 is a prerequisite for peroxisomal regulation and served as a critical signaling in this study.Next, we further explored whether PEX3 is valuable in myocardial regeneration". 1. Shimozawa N, Suzuki Y, Zhang Z, Imamura A, Ghaedi K, Fujiki Y, Kondo N. Identification of PEX3 as the gene mutated in a Zellweger syndrome patient lacking peroxisomal remnant structures.Hum Mol Genet.2000 Aug 12;9( 13

Ad5 PEX3 overexpression suggests it is cardioprotective, while AAV9 PEX3 overexpression suggests it promotes positive regeneration. Are these linked?
Response: Thanks for the valuable comments.We apologize for the lack of a uniform description in our manuscript.Cardiac protection and repair after an injury is a grand scope, including cardiomyocyte proliferation, inflammation regulation, neovascularisation and interstitial remodelling 1 .In our research, transfection of Ad5: cTNT-PEX3 could promote the proliferation of neonatal mice primary cardiomyocytes in vitro.In vivo, transfection of AAV9: CTNT-PEX3 could promote the proliferation of cardiomyocytes and improve cardiac function after MI.The above results indicated that PEX3 promoted cardiomyocyte proliferation and cardiac repair.We have revised the expression accordingly in the manuscript.1. Uygur A, Lee RT.Mechanisms of Cardiac Regeneration.Dev Cell.2016 Feb 22;36(4):362-74.PMID: 26906733; PMCID: PMC4768311. 5. I assume AR in line 247 means apical resection?I find it strange that this was the choice to induce heart injury.The neonatal LAD model (which is later used in the manuscript in adults) would be more appropriate to conduct the experiments since this model better induces the hypoxia/ischemia conditions seen following MI.
Response: Thanks.AR refers to apical resection, which is a common animal model for studying myocardial regeneration in neonatal mice [1][2][3] .LAD ligation of neonatal mouse hearts assisted by somatoscopy requires a very high surgical technique, and this operation is not yet established in our laboratory; the next, we will try this model in future studies.Thank you for this stimulating and helpful comment.1. Ji X, Chen Z, Wang Q, Li B, Wei Y, Li Y, Lin J, Cheng W, Guo Y, Wu S, Mao L, Xiang Y, Lan T, Gu S, Wei M, Zhang JZ, Jiang L, Wang J, Xu J, Cao N. Sphingolipid metabolism controls mammalian heart regeneration.Cell Metab.2024 Apr 2;36( 4 6.increased fibrosis in Pex3-KO mentioned from line 263 onwards needs to be explored more.How is PEX3 and to a broader degree peroxisomes mechanistically affecting fibrosis?Are the fibroblasts being activated?How do peroxisomes interact with fibroblasts when the model presented is a cardiomyocyte specific KO?Is the increase in fibrosis coming from increased immune cell activity/activation? How this links to the proliferation data is also not clear Response: Thanks for the insightful comments.As suggested, we performed immunofluorescence co-localization experiments of PEX3 and non-cardiomyocytes, and the results showed that PEX3 was mainly activated/inhibited in cardiomyocytes after neonatal/adult myocardial injury, but not in fibroblasts/endothelial cells/macrophages (Figure 1E-F and Supplementary Figure 2A-B).Therefore, we believe that when Pex3-KO mostly affects cardiomyocytes, future studies are needed to explore the above issued comments.We added the related study limitation now.It reads (Page 36, line 1003-1008): "Although Pex3-KO might largely affect the fate of CMs, the exact impacts and mechanisms of Pex3-KO or PEX3 overexpression, on fibroblast, the role of non-CMs including immune cells on fibrosis, their impact on regenerative capacity, the impact on non-CMs as well as the interaction on fibrosis, CM proliferation as well as myocardial regeneration are not observed in this study and warrant further experimental studies".7. similarly, in the PEX3 overexpression in adult MI model, the data suggests the scar is being removed.Is this really the case?If so how is this achieved given that AAV9 delivery is generally cardiomyocyte specific.Are other cell types such as fibroblasts or immune cells involved?
Response: Thanks for the fair comments.This is an assay of great value in previous studies in the field of cardiomyocyte proliferation as well as myocardial regeneration 1-2 .In our study, we focused on the impact of overexpression of PEX3 on proliferative replenishment of cardiomyocytes and their role in the reduction of fibrosis after myocardial injury, while their impact on regenerative capacity, the impact on non-cardiomyocytes as well as the interaction on fibrosis, cardiomyocyte proliferation as well as myocardial regeneration remains unknown and need to be verified in future studies.This study limitation is added in the revised manuscript now (Page 36, line 1003-1008)

echo analysis to assess function at 28dpi is line 294 is rather early to state a functional recovery, particularly when the scar is still present. Please provide later timepoints if the authors want to keep this statement or tone down
Response: Thank you very much for your professional advice.We have modified the presentation of the 28dpi echocardiography results in the appropriate section of the manuscript to achieve more accuracy.It reads (Page 26, line 709-710): "echocardiography results revealed that PEX3 overexpression improved cardiac function at 28dpi".9. it is unclear if the authors are trying to establish a direct link between proliferation and fibrosis following PEX3 modulation.The text in some places suggests it is while not in other places.Please be clearer and if a direct link is suggested, please provide the data to support these claims.
Response: Thanks for this comment.We apologize for the unclear presentation.Our results showed that modulating PEX3 expression could affect the proliferative capacity of cardiomyocytes after myocardial injury, which might lead to differences in regenerative repair or scar filling after myocardial injury.A solid direct link could not be established based on available data, since other factors might also contribute to the proliferation and fibrosis changes following PEX3 modulation.We have modified the manuscript accordingly.It reads (Page 25, line 679-680): "Moreover, Masson staining showed that PEX3 knockdown inhibited myocardial regeneration"; (Page 26, line 710-712): "Additionally, Masson staining demonstrated that PEX3 overexpression significantly reduced the infarct size"; (Page 27, line 733-734): "PEX3 knockdown significantly increased the infarct size at 28dpi"; (Page 28, line 771-772): "In addition, Masson staining showed that plasmalogen supplementation could reduce the infarct size at 28dpi".

ITGB3 appears to be downstream of PEX3, what do the peroxisomes look like in ITGB3 overexpression/knockdown? Is the PEX3/ITGB3 interaction direct or only indirect via plasmalogens
Response: Thanks for the important comments.Yes, ITGB3 is a downstream regulatory target of PEX3.Our experiments in Result 8 (Figure 7 and Supplementary Figure 10-11) showed that PEX3 regulates the plasma membrane localization of ITGB3 and functions as a signal transducer by affecting acetylated phospholipid metabolism, thus regulating ITGB3.To further clarify that ITGB3 is a downstream regulatory target of PEX3, we further examined the changes in the expression of PEX3 and PMP70 after intervening with ITGB3, and the results showed that the alteration of ITGB3 did not affect the expression of PEX3 and PMP70 (Supplementary Figure 10C-F).The text has now been rephrased to present this point more clearly in the revised manuscript.
Response: Thanks for the comment.As suggested, we detected the expression changes of PMP70 after intervening in ITGB3, and the results showed that intervention in ITGB3 did not affect the expression of PEX3 and PMP70 (Supplementary Figure 10D-F Response: Thank you for bringing up the issue of unexplained abbreviations.In our manuscript, "dps" stands for days post sham, "dpr" stands for days post apical resection, "AR" stands for apical resection, "DCF" stands for 2',7'-Dichlorofluorescein, and "MOI" stands for multiplicity of infection.We have compiled a list of all the abbreviations used in the study now (Page 4-5, line 59-115).
2. Please specify more clearly what is P7/6dps, P7/6dpr, P56/6dps/P56/6dpi.These abbreviations were not defined anywhere in the main text and i had to go hunting in an unrelated figure legend to figure out what the authors were talking about.Please rename these abbreviations to something that is more easy for the reader to understand.There is already so much abbreviations for other names in the text that the author's message gets lost Response: Thank you for raising this concern, and we apologize for the lack of clear explanations for these abbreviations.In our manuscript, "P7/6dps" represents postnatal day 7, specifically 6 days post sham operation."P7/6dpr" represents postnatal day 7, which is 6 days post apical resection performed on postnatal day 1.Similarly, "P56/6dps" and "P56/6dpi" respectively represent heart tissue samples from the control group at postnatal day 56, 6 days post sham operation, and heart tissue samples from the myocardial infarction group at postnatal day 56, 6 days post-myocardial infarction.We have also compiled a separate list of all the abbreviations used in the study to minimize any unnecessary misunderstandings (Page 4-5, line 59-115).
Sun et al. aims to identify the role of the peroxisome regulator, PEX3, in myocardial regeneration.The authors observed an increase in RNA and protein levels of PEX3 during regeneration.They found that deletion of pex3 reduces postnatal cardiomyocyte proliferation, while overexpression of Pex3 promotes this proliferation in vitro.Importantly, they demonstrate that Pex3 deletion impedes cardiomyocyte proliferation and neonatal heart regeneration following apical resection.Additionally, in vivo overexpression of Pex3 via AAV9 enhances adult cardiomyocyte proliferation, decreases scar size, and improves cardiac function post-myocardial infarction (MI).To explore the mechanisms behind Pex3's role in myocardial regeneration, the authors conducted lipidomic analyses and discovered a reduction in plasmalogen levels in Pex3 knockout mice.Supplementation of plasmalogen improved the regenerative response in these mice.Further investigations revealed that plasmalogen regulates the localization of ITGB3 in the plasma membrane, which in turn mediates AKT signaling involved in the regenerative effects of Pex3.Therefore, the authors conclude that Pex3 facilitates cardiomyocyte proliferation and regeneration through the modulation of plasmalogen metabolism, which affects ITGB3 and AKT signaling.This is a comprehensive study that provides new insights into the role of Pex3 and the peroxisome during heart regeneration.A few points can be addressed which would strengthen the conclusions of this study.